1. Field of the Invention
The present invention relates generally to the fields of biophysics, tissue regeneration, tissue culture, and neurobiology. More specifically, the present invention relates to the use of an electromagnetic field, and preferably, a time varying electromagnetic field, for potentiation of or controlling the growth of biological cells and tissue, such as mammalian tissue. More specifically, the present invention relates to the use of an electromagnetic field for controlling the growth of neural cells and tissues. The preferred embodiment utilizes two-dimensional conducting plate electrodes and may be applied to conventional, two dimensional tissue cultures or to three-dimensional cultures. Three dimensional cultures may be achieved in actual microgravity or by rotating wall vessel technology which simulates the physical conditions of microgravity, and in other, conventional three-dimensional matrix based cultures. The electromagnetic field, preferably a time varying electromagnetic field, is achieved in the vicinity of the electrode by passing, through the electrode, a time varying current.
2. Description of the Related Art
Growth of a variety of both normal and neoplastic mammalian tissues in both mono-culture and co-culture has been established in both batch-fed and perfused rotating wall vessels (1-2), and in conventional plate or flask based culture systems. In some applications, growth of three-dimensional structure, e.g., tissues, in these culture systems has been facilitated by support of a solid matrix in the form of biocompatible polymers and microcarriers. In the case of spheroidal growth, three-dimensional structure has been achieved without matrix support (3-6). NASA rotating wall tissue culture technologies have extended this three dimensional capacity for a number of tissues and has allowed the tissue to express different genes and biomolecules. Neuronal tissue has been largely refractory, in terms of controlled growth induction and three dimensional organization, under conventional culture conditions. Actual microgravity, and to a lesser extent, rotationally simulated microgravity, have permitted some enhanced nerve growth (Lelkes et al). Attempts to electrically stimulate growth have utilized static electric fields, static magnetic fields, and the direct passage of current through the culture medium, though not the induction of a time varying electromagnetic field in the culture region.
Neuronal tissue comprises elongated nerve cells composed of elongated axons, dendrites, and nuclear areas. Axons and dendrites are chiefly responsible for transmission of neural signals over distance and longitudinal cell orientation is critical for proper tissue formation and function. The nucleus plays the typical role of directing nucleic acid synthesis for the control of cellular metabolic function, including growth. In vivo, the neuronal tissue is invariably spatially associated with a system of feeder, or glial, cells. This three dimensional spatial arrangement has not been reproduced by conventional in vitro culture. Investigators, Borgens RB et al, and others, have utilized static electric fields in an attempt to enhance nerve growth in culture. (Valentini et al) with some success to either alter embryonic development or achieve isolated nerve axon directional growth. However, actual potentiation of growth or genetic activity causing such, have not been achieved. Mechanical devices intended to help grow and orient three dimensional mammalian neuronal tissue are currently available. Fukuda et al. (7) used zones formed between stainless steel shaving blades to orient neuronal cells or axons. Additionally, electrodes charged with electrical potential were employed to enhance axon response. Aebischer (8) described an electrically-charged, implantable tubular membrane for use in regenerating severed nerves within the human body. However, none of these devices utilize channels of cell-attractive material, neither do they apply a time varying electromagnetic field, or a static electrical or magnetic field. Additionally, no use is made of simulated or actual microgravity techniques for pure neuronal, or mixed, neuronal and feeder cell cultures. The prior art is deficient in its lack of effective means for growing three dimensional mammalian neuronal tissue in the proximity of, or directly upon the surface, of a current carrying electrode (which may be bioattractive and directly adherent to the cells). Furthermore, the use of a time varying current to induce a corresponding time varying electromagnetic field, in the vicinity of the growing culture, to potentiate or spatially direct cell growth is not part of the prior art.
The present invention relates to a system and method for culturing biological cells, such as mammalian cells, within a culture medium. The cells are exposed to an electromagnetic field, which, in the preferred embodiment, is a time-varying electromagnetic field. In the preferred embodiment, this field is generated by a conductive electrode, adjacently spaced from the incubating cells, carrying a time varying electrical current. The electrode, in one case, is in direct galvanic contact with the culture media and cells, and in another case, it is placed external to the culture apparatus in a galvanically isolated condition. Preferably, a 10 hertz square wave of 1-6 milliamperes, and with nearly zero time average, is passed through the electrode, suitably from corner to opposite comer of a square metallic conductive plate. The cells, such as neurons in this case, were, in one embodiment, grown directly on the electrode surface, composed of a biocompatable material. In another embodiment, the cells were grown within a container under the influence of a time varying electromagnetic field from an electrode external of and adjacent to the container, galvanically isolated from the media and culture within the container.
The growing cells may actually be attracted and trophically supported by more supportive electrode material or coatings. Furthermore, channels may be incorporated in the culture vessel and lined with growth substrate which may be electrically conductive. In one embodiment, a time varying electromagnetic field is induced in the region of the channel by passing the time varying current through a conductor placed along the channel. This arrangement will further direct growth by the combined effect of the field and trophic materials.
In the preferred embodiment, the presence of the time varying electromagnetic field potentiates the growth of nerve and other tissue. The time varying field may be induced by either: 1) a time varying current within a conductor, or 2) a time varying voltage between fixed conductors. In one embodiment, for example, the culture is placed nearby a conductor through which a time varying current is passed, or between parallel plates upon which a time varying voltage is applied. In both cases, a time varying electromagnetic field results within the area of interest, i.e., in the region of the cell culture.
The system and process are utilized in combination with known tissue culture processes to produce enhanced cell growth, directed cell growth, and tissue formation and organization.
As will be understood from the description to follow, the system is operable to up or down regulate the activity of specific genes. In general, growth promoting genes are up regulated and growth inhibitory genes are down regulated. The effect is shown to persist for some period after termination of the applied time varying field. This persistent, growth promoting effect subsides after a period of some days, and the cells return to a growth state characterized by controls, having never been exposed to the fields. This is beneficial in certain applications, in that medical applications for clinical medical care, i.e. nerve regeneration, are therefore safer than if the xe2x80x9cpseudo transformedxe2x80x9d state persisted. The set of gene transformations, associated with the time varying electromagnetic field, also promote the ability of the growing tissue to adhere and thrive on substrates by the induction of genes leading to the secretion of extracellular materials favorable to the tissue microenvironment.
Several methods of producing the time varying electromagnetic field in the vicinity of the living tissue culture are encompassed. In one embodiment, an array of conductive current carrying elements (or voltaic electrodes) are arranged so as to intensify or focus the time varying electromagnetic (EM) field onto the culture. Each embodiment is characterized by a method for application of the time varying field to the target tissue, such as neuronal, for stimulation of growth, or repair or induction of changes in gene activity patterns. The term xe2x80x9cfield generatorxe2x80x9d is used herein to represent these various embodiments for generating the time varying electromagnetic fields. In its simplest form, it is a conductive electrode, placed near the target cells, through which current is directed from a controlled waveform current source.
As suggested above, in one embodiment, the field generator is in the form of a conductive channel mounted on or embedded in a disc of biocompatable material. (FIG. 11) One or more of these discs may be then placed within a rotational bioreactor so as to obtain the beneficial culture conditions associated therewith. The combination of the stimulatory electromagnetic field with the rotational environment, known to permit morphological expression beyond conventional culture, is particularly effective. This is because the induced pattern of growth enhancing genes is permitted to be ultimately expressed, as cell growth and tissue formation, without mechanical inhibition from the culture apparatus. Also the inherent growth advantages well known in the rotational systems is synergistic with the growth stimulation derived from the time varying electromagnetic field. The conditions may be further optimized by utilizing actual microgravity, in space. In this application, mechanical rotation of the cell culture vessel is not required but may be utilized to achieve mixing and sufficient mass transfer to sustain a healthy culture. Other forms of mixing may be introduced as necessary to achieve adequate mass transfer for each embodiment.
In one embodiment, illustrated in FIGS. 10 and 11, slip-ring contacts or their equivalents are electrically connected to the ends of the channels, and an external power source is provided for applying the time varying electrical current defining the waveform through the channels. In another embodiment, the channel consists of a pair of parallel, mutually spaced conductors across which a time varying voltage is applied. This also achieves the time varying electromagnetic field but restricts it to the region between the parallel electrodes, which is advantageous for directing localized growth according to a desired physical pattern. The present invention also relates to a system and method for culturing primarily two dimensional mammalian cells facilitated by a time varying electromagnetic field. The electrodes may either be in direct galvanic contact or galvanically isolated from the target cells. The present invention provides a strategy to re-engineer nerve tissue and myoneural junctions and can be used medically for axonal regeneration.
In one embodiment of the present invention, there is provided a system for growing three dimensional mammalian cells, comprising a rotating wall vessel containing a cell-rich medium and a formed cell growth substrate. A time varying electromagnetic field is applied to enhance tissue growth which may occur on a shaped substrate. The electromagnetic field may be generated by means such as by directing the current waveform directly through a conductive substrate (or substrate layer) or by projecting the field from an external antenna, or electrode adjacent to and spaced from the medium, the spacing being sufficiently small relative to the strength of the electromagnetic field to induce effectual levels of electromagnetic field within the medium, in accordance with the particular application. A time varying electromagnetic field may be emitted from a nearby plate or other suitable xe2x80x9cantennae,xe2x80x9d or a time varying voltage may be applied across suitable electrodes (such as plates) to produce the time varying electromagnetic field. The field generation system may either be rotating with the vessel or fixed, and spaced from, the rotating vessel. The rotating wall vessel can be a rotating wall perfused vessel or a rotating wall batch-fed vessel.
The time varying electromagnetic field is advantageously produced by a varying electrical potential in the form of a square wave having a frequency of approximately ten cycles per second. In one embodiment, a current of about ten milliamps, conducted between opposite comers of a metallic conductor, produces a stimulatory time varying electromagnetic field extending several centimeters from the plate surface. In practice, the range of frequency and oscillating electromagnetic field strength is a parameter which may be selected to for achieving the desired stimulation of particular tissues, cells, or genes, and for providing the appropriate amount of up/down regulation of these genes.
In one embodiment of the present invention, the cell growth susbtrates or carriers are spherical disks containing multiple parallel channels (FIG. 10) which are coated with a bioattractive material. The bioattractive material has a longitudinal axis across which the time varying electrical potential is applied and through which a time varying current is conducted. The mammalian cells adhere to the bioattractive material and are free to orient, as they grow. Representative bioattractive materials include titanium, zirconium and platinum.
The class of mammalian cells preferably is selected from the group consisting of neuronal cells, normal human neuronal progenitor cells (NHNP), and a cell responding to the time varying electromagnetic field. It will be understood by those of ordinary skill in the art that the teachings of the present invention apply to other cell types.
In another embodiment of the present invention, there is provided a method of culturing mammalian cells in the claimed system, comprising the steps of inoculating the cells into the vessel containing a culture medium, rotating the vessel to enhance the proliferation of the cells and, in one embodiment, to initiate the attachment of the cells to microcarrier spheres or beads suspended within the culture medium, applying a time varying electromagnetic field to the culture medium, cells, and cell carriers, and measuring the growth of the cells. Preferably, the vessel is rotated at a speed from about 2 RPM to 30 RPM, and the time varying electromagnetic field is generated by a time varying current passed through a conductor with RMS value of about 1 to 1,000 ma. In one embodiment, a range of about 1 mA to 6 mA is used.
In still another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells, comprising a petri dish containing a cell culture medium and an electrode placed in the center of the petri dish. In this embodiment, the electrode serves as the field generator. Preferably, the neural cells are applied directly on the electrode. As a result, the neutral cells exhibit accelerated growth.
In yet another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells further comprising a slide placed on the electrode. Preferably, the neural cells are applied, e.g., bubbled, on the slide instead of directly contacting the electrode, and preferably, the current producing the waveform is applied at a strength range of from about 1 mA to about 100 mA, and, in one embodiment, suitably from about 1 mA to 6 mA.
In still another embodiment of the present invention, there is provided a method of treating an individual having diseased neuronal cells, comprising the steps of growing neuronal cells in the claimed two- or three- dimensional systems and transplanting the neuronal cells into the individual. Such diseases include Parkinson""s disease, diseases of neuromuscular junction and Alzheimer""s Disease. Neural trauma can also be treated in same methodology.
In yet another embodiment of the present invention, the time varying electromagnetic field (or electrical potential) induces cellular response including cellular control of growth and differentiation at gene level. Preferably, the cellular control of growth and differentiation is to suppress or enhance growth regulatory functions at gene level. Still preferably, the gene is associated with increased tissue and cell proliferation.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.